Field of the Invention
This invention relates to a chimeric protein made from a combination of thionin and D4E1. This invention also relates to genetically altered plants that can express this chimeric protein, and the use of the chimeric protein to protect plants against bacterial infections.
Description of Related Art
Plants have developed multiple defense mechanisms to combat invading pathogenic microorganisms. For example, plants will synthesize antimicrobial compound such as antimicrobial peptides (AMPs), thionins and defensins. See, Broekaert, et al., Critical Review in Plant Sci. 16:297-323 (2007). Many AMPs have been identified from various organisms. AMPs are short peptides with broad spectrum antimicrobial activity against bacteria and fungi. AMPs can damage a pathogen's cell membrane by inhibiting chitin synthase or β-D-glucan synthase. See, DeLucca and Walsh, Antimicrobial Agent and Chemotherapy 43:1-11 (1999). D4E1, one such antimicrobial peptide which is also a synthetic peptide, has a β-sheet conformation in solution and during interaction with cell membranes which results in lytic activity. See, DeLucca, et al., Can. J. Microbiol. 44:514-520 (1998). In order to optimize the activity against target pathogens, chimeric protein constructions, with or without modification of amino acid sequences, have been designed including the anti-fungal and anti-bacterial lactoferricin B derivatives (Marcos, et al., Annu. Rev. Phytopathol. 46:273-301 (2008), antifungal cecropin A and cecropin A-mellitin derived peptides (Monroc, et al., Peptides 27:2567-2574 (2006); Monroc, et al., Peptides 27:2575-2584 (2006); and Cavallarín, et al., Mol. Plant Microbe Interact. 11:218-227 (1998)), and bactericidal cyclic decapeptide BPC194 series (Monroc, et al., Peptides 27:2567-2574 (2006); and Monroc, et al., Peptides 27:2575-2584 (2006)), and antifungal hexapeptide PAF26 (López-García, et al., Mol. Plant Microbe Interact. 13:837-846 (2000); and López-García, et al., Appl. Environ. Microbiol. 68:2453-2460 (2002).
Many studies have reported that the expression of naturally occurring peptides and their analogs confer resistance to pathogens in transgenic plants including Arabidopsis (Lee, et al., Plant Physiol. 148:1004-1020 (2008)), tobacco (Huang, et al., Phytopathology 87:494-499 (1997); Jaynes, et al., Plant Sci. 89:43-53 (1993); and Cary, et al., Plant Sci. 154:171-181 (2000)), Chinese cabbage (Jung, et al., Plant Biotechnol. Rep. 6:39-46 (2012)), rice (Coca, et al., Planta 223:392-406 (2006), and Imamura, et al. Transgenic Res. 19:415-424 (2010)), cotton (Rajasekaran, et al., Plant Biotechnol. J. 3:545-554 (2006)), tomato (Alan, et al., Plant Cell Rep. 22:388-396 (2006), and Alan, et al., Plant Cell Rep. 22:388-396 (2006)), potato (Osusky, et al., Transgenic Res. 13:181-190 (2004)), pear (Reynoird, et al., Plant Sci. 149:23-31 (1999)), banana (Chakrabarti, et al., Planta 216:587-596 (2003)), and hybrid poplar (Mentag, et al., Tree Physiol. 23:405-411 (2003)).
Compared to naturally produced peptides, synthetic peptides such as D4E1 show rapid biocontrol or biostatic ability against various fungal and bacterial pathogens at low concentrations and can be designed to be non-toxic to mammalian and other animal cells (Jaynes, et al., Peptide Res. 2:157-160 (1989). In addition, synthetic peptides are generally designed to resist degradation by fungal and plant proteases and show target specificity and increased efficacy (Broekaert, et al. (2007), and Montesinos, E, FEMS Microbio. Lett. 270:1-11 (2007)). Haemolytic activity of synthetic peptide D4E1 was found to be very low (Jaynes, et al., Plant Sci. 89:43-54 (1993)). It has been demonstrated that the pure synthetic peptide D4E1 is inhibitory to growth of about 20 bacterial and fungal phytopathogens (Rajasekaran, et al., J. Agric. Food Chem. 49:2799-2803 (2001)). In addition, transgenic tobacco plants expressing D4E1 demonstrated a significant reduction in fungal growth in vitro and in planta (Cary, et al., Plant Sci. 154:171-181 (2000)). A 50% to 90% reduction in the viability of Fusarium verticillioides and Verticillium dahliae was reported when spores were incubated in crude protein extracts of D4E1-transformed cotton compared to extracts from GUS-transformed cotton (Rajasekaran, et al., Plant Biotechnol. J. 3:545-554 (2005)). D4E1 is effective in killing Chlamydia trachomatis in-vitro (Ballweber, et al., Antimicrobial Agents and Chemotherapy 46:1, 34-41 (2002)). Purified D4E1 is highly effective at killing Agrobacterium tumefaciens, Sinorhizobium meliloti, and Xanthomonas citri ssp. citri, but shows little hemolysis of porcine erythrocytes (Stover, et al., J. Amer. Soc. Hort. Sci. 138:142-148 (2013)).
Thionins from barley leaf are toxic to phytopathogenic bacteria and fungi in-vitro (Bohlmann, et al., EMBO J. 7:1559-1566 (1988); Molina, et al., Plant Sci. 92:169-177 (1993)). Thionins are small basic peptides containing between approximately 44 and approximately 47 amino acids and contain a conserved cysteine-rich domain with toxic and antimicrobial properties. Thionins are classified into two groups, a/b-thionins and c-thionins, based on their 3-D structure (Pelegrini and Franco, Int. J. Biochem. Cell. Bio. 37:2239-2253 (2005)). Thionins are postulated to induce the opening of pores on the pathogen-cell membranes, allowing escape of potassium and calcium ions from pathogens' cell(s) (Pelegrini and Franco (2005); Oard, S. V., Biochim. Biophys. Acta 1808:1737-1745 (2011)). It has been showed that a thionin gene from barley seed increased resistance to Pseudomonas syringae when overproduced in transgenic tobacco plants (Carmona, et al., Plant J. 3:457-462 (1993)). Overexpression of the Arabidopsis thionin thi2.1 gene in Arabidopsis plants resulted in enhanced resistance to Fusarium oxysporum (Epple, et al., Plant Cell 9:509-520 (1997). Transgenic rice plants overproducing oat thionin displayed enhanced resistance to the bacterial diseases caused by Burkholderia plantarii and B. glumae (Iwai, et al., Mol. Plant Microbe Interact. 15:837-846 (2002)). Likewise, transgenic sweet potato overproducing barley α-hordothionin had increased resistance to black rot disease caused by Ceratocystis fimbriata (Muramoto, et al., Plant Cell Rep. 31:987-97 (2012)).
To improve the efficacy of these anti-microbial agents, plants transformed to express chimeric proteins containing two different proteins or peptides have been assessed. In a recent study, grape plants were transformed to express a chimeric protein containing human neutrophil elastase and cecropin B. These transgenic grape plants had resistance against Xylella fastidiosa ssp. fastidiosa which causes Pierce disease in grapevines. See, Dandekar, et al., Proc. Nat. Acad. Sci. 109:10 3721-3725 (2012). These finding are important because X. fastidiosa is a Gram-negative bacterial pathogen with a wide range of plant hosts of economic importance.
Huanglongbing (HLB) (also called “citrus greening”) and citrus canker cause serious diseases that threaten the Florida citrus industry. The causative agents of HLB are Candidatus Liberibacter africanus (CLaf), Candidatus Liberibacter asiaticus (CLas), and Candidatus Liberibacter americanus (CLam). The bacteria are transmitted from plant to plant via Asian citrus psyllid (Diaphorina citri), the African citrus psyllid (Trioza erytreae), and, perhaps, other hemipteran insects that feed from citrus plants' vascular tissue. Citrus canker is caused by Xanthomonas citri ssp. citri (Xcc). Xcc are spread by wind and perhaps insects and enter into citrus plants via wounds or other openings in the bark. These diseases are devastating the citrus industry in Florida because of no effective treatment currently exists. Furthermore, both diseases and the causative pathogens are spreading to other parts of the U.S. and other citrus-producing countries.
A need exists for a method to prevent and/or treat these diseases in citrus plants. A need also exists to prevent and/or treat bacterial diseases in plants. This invention involves genetically altered citrus plants that can produce a chimeric protein having broad-spectrum antimicrobial activity against gram-negative bacteria which can confer resistance to both citrus greening and canker diseases in citrus plants. This invention also involves other genetically altered plants expressing this chimeric protein and that have resistance to diseases caused by bacteria.